The success in generating murine monoclonal antibodies rests on the efficient and selective fusion of antigen-stimulated B cell blasts with a murine myeloma cell line followed by selection of stable antibody producing hybrids (Kohler & Milstein 1975). FR2817875 describes a modified version of this protocol where prior to immortalisation, B lymphocytes are induced to differentiate by a non-specific activating system and a cytokine. The B cell blasts may be taken from the spleen or lymph nodes. However, the difficulty in obtaining antigen-stimulated B cell blasts and the lack of suitable fusion partners has hampered this approach in the human system.
As an alternative approach to making human antibodies, Epstein Barr Virus (BV) has been used to immortalize human (and primate) B cells producing specific antibodies. The EBV method has been described in several publications since 1977 (Rosen et al. 1977; Steinitz et al. 1977; Steinitz et al. 1980; Kozbor & Roder 1981; Lundgren et al. 1983; Rosen et al. 1983; Steinitz et al. 1984; Lanzavecchia 1985). B cells are immortalized by infection with EBV and growing clones secreting specific antibodies are selected. The method does not require antigenic boost, since EBV immortalizes also resting human B cells. However, the EBV-based method has several limitations, namely the low efficiency of immortalization, the low cloning efficiency of EBV-immortalized B cells, the slow grown rate and, in some cases, low antibody production. U.S. Pat. No. 5,499,7764 describes a method of improving the growth rate of the EBV immortalised cells comprising transfecting EBV infected B cells with activated c-myc DNA. This confers on the cells the ability to grow in semi-solid media and to grow in hosts such as rats and mice. WO95/13089 describes the use of GM-CSF and IL-3 to stimulate the release of antibody by B cells. Boimkanim et al. (U.S. Pat. No. 5,798,230) have overcome the problem of low production of antibodies by inactivating EBNA2. However, this does not solve the problem of low efficiency of immortalization. To circumvent these problems some authors carried out an enrichment for antigen-specific B cells before EBV immortalization using for instance biotinylated soluble antigens (Casali et al. 1986). Others proposed the fusion of the EBV-immortalised cells with mouse myelomas or human-mouse heteromyelomas to exploit the higher growth rate and Ig secretion of the hybrids (Kozbor et al. 1982; Bron et al 1984; Thompson et al. 1986). Claims that the cloning efficiency could be increased by cell-derived growth factors such as thioredoxin have been made in a publication (Ifversen et al. 1993), but these results have neither been confirmed nor utilized, even by the same authors. In conclusion, although the EBV method has in principle some advantages, it has been abandoned because of the low efficiency of immortalization and cloning.
Another reason why the EBV method has become obsolete is that alternative approaches for making human or human-like monoclonal antibodies became available through genetic engineering. These include the humanization of murine antibodies, the isolation of antibodies from libraries of different complexity and the production of hybridomas using the classical method in mice transgenic for human Ig loci (the “xeno-mouse”). The literature on these alternative approaches is not reviewed here since is not directly relevant to the present invention. However, it is worth considering some limitations of these methods. Humanization of murine monoclonal antibodies is a laborious and incomplete procedure. Random antibody libraries represent an unbiased repertoire and can therefore be used to select antibody specificities against highly conserved antigens, but lead to antibodies of low affinity. Libraries selected from antigen primed B cells are enriched for a particular specificity, but do not preserve the original VH-VL pairing and generally lead to antibodies that have lower affinity for the antigen than those present in the original antibody repertoire. The impact of this technology has been limited. In contrast the xeno-mouse can be efficiently immunized against an antigen of choice (especially if this is a human antigen), but this system shares with the classical murine hybridoma technology the limitation that the antibodies are selected in a species other than human. Therefore these methods are not suitable to produce antibodies with the characteristics of those produced in the course of a physiological human immune response. This applies to the antibody response to human pathogens including HIV, the four Plasmodium species that cause malaria in humans (P. falciparum, P. vivax, P. malariae and P. ovale), human hepatitis B and C viruses, Measles virus, Ebola virus etc. (for an exhaustive list see Fields et al. 1996). It also applies to antibody responses to environmental allergens generated in allergic patients, to tumour antigens generated in tumour bearing patients and to self antigens in patients with autoimmune diseases.
There is therefore a need for an efficient method of production of human monoclonal antibodies that have been selected in the course of the natural immune response.